Deglacial volcanism and reoxygenation in the aftermath of the Sturtian Snowball Earth.

The Cryogenian Sturtian and Marinoan Snowball Earth glaciations bracket a nonglacial interval during which Demosponge and green-algal biomarkers first appear. To understand the relationships between environmental perturbations and early animal evolution, we measured sulfur and mercury isotopes from the Datangpo Formation from South China. Hg enrichment with positive Δ199Hg excursion suggests enhanced volcanism, potentially due to depressurization of terrestrial magma chambers during deglaciation. A thick stratigraphic interval of negative Δ33Spy indicates that the nonglacial interlude was characterized by low but rising sulfate levels. Model results reveal a mechanism to produce the Δ33S anomalies down to -0.284‰ through Rayleigh distillation. We propose that extreme temperatures and anoxia contributed to the apparent delay in green algal production in the aftermath of the Sturtian glaciation and the subsequent reoxygenation of the iron-rich and sulfate-depleted ocean paved the way for evolution of animals.

Palaeoecology of the Hiraiso Formation (Miyagi Prefecture, Japan) and implications for the recovery following the end-Permian mass extinction.

The Hiraiso Formation of northeast Japan represents an important and under-explored archive of Early Triassic marine ecosystems. Here, we present a palaeoecological analysis of its benthic faunas in order to explore the temporal and spatial variations of diversity, ecological structure and taxonomic composition. In addition, we utilise redox proxies to make inferences about the redox state of the depositional environments. We then use this data to explore the pace of recovery in the Early Triassic, and the habitable zone hypothesis, where wave aerated marine environments are thought to represent an oxygenated refuge. The age of the Hiraiso Formation is equivocal due to the lack of key biostratigraphical index fossils, but new ammonoid finds in this study support an early Spathian age. The ichnofossils from the Hiraiso Formation show an onshore-offshore trend with high diversity and relatively large faunas in offshore transition settings and a low diversity of small ichnofossils in basinal settings. The body fossils do not, however, record either spatial or temporal changes, because the shell beds represent allochthonous assemblages due to wave reworking. The dominance of small burrow sizes, presence of key taxa including Thalassinoides, Rhizocorallium and Holocrinus, presence of complex trace fossils, and both erect and deep infaunal tiering organisms suggests that the benthic fauna represents an advanced stage of ecological recovery for the Early Triassic, but not full recovery. The ecological state suggests a similar level of ecological complexity to late Griesbachian and Spathian communities elsewhere, with the Spathian marking a globally important stage of recovery following the mass extinction. The onshore-offshore distribution of the benthic faunas supports the habitable zone hypothesis. This gradient is, however, also consistent with onshore-offshore ecological gradients known to be controlled by oxygen gradients in modern tropical and subtropical settings. This suggests that the habitable zone is not an oxygenated refuge that is only restricted to anoxic events. The lack of observed full recovery is likely a consequence of a persistent oxygen-limitation (dysoxic conditions), hot Early Triassic temperatures and the lack of a steep temperature/water-depth gradient within the habitable zone.

Devonian to Carboniferous continental-scale carbonate turnover in Western Laurentia (North America): upwelling or climate cooling?

The Devonian to Carboniferous (DC) transition coincided with a green-to-ice house climatic shift, anoxia, disappearance of lower latitude carbonate banks, and turnover from warm-to-cool water carbonate factories. In western Laurentia, the switch to carbonate factories dominated by cool-water biota was contemporaneous with a tectonically driven palaeogeographic change. To investigate this depositional shift and infer the relative impact of climate vs tectonics, a continental-scale sedimentological and geochemical study was conducted on twelve stratigraphic sections of DC strata from western Canada to southern Nevada (USA). The spatial-temporal distribution of microfacies records the turnover from [i] a Famennian lime mud-rich, shallow warm-water carbonate ramp with low sedimentation rates, mesotrophic conditions and tabular geometry to [ii] Tournaisian to Viséan lime mud-depleted and grainstone dominated cool-water carbonate ramp with anomalous high sedimentation rates, oligotrophic conditions and a pronounced slope. Positive excursions of δ 18Ocarb (+ 2‰ V-PDB) and δ 13Ccarb (+ 4‰ V-PDB) of Lower Mississippian carbonates likely correspond to the first cooling peak of the Carboniferous-Permian icehouse climate, following carbon withdrawal during black shale deposition during the late Famennian and early Tournaisian. However, late Tournaisian return of photozoan elements and their persistence throughout the Viséan suggests that warmer surface water existed, revealing a decoupling of the lower latitude ocean and the atmosphere. Shoaling of the thermocline was likely a result of cold-water upwelling along an open coast, as the Antler orogen no longer provided an oceanic obstruction to the west. This study shows that carbonate platforms are more susceptible to regional changes than global shifts. Supplementary Information: The online version contains supplementary material available at 10.1007/s10347-022-00653-4.

Microbial Functional Diversity Correlates with Species Diversity along a Temperature Gradient.

Microbial community diversity is often correlated with physical environmental stresses like acidity, salinity, and temperature. For example, species diversity usually declines with increasing temperature above 20°C. However, few studies have examined whether the genetic functional diversity of community metagenomes varies in a similar way as species diversity along stress gradients. Here, we investigated bacterial communities in thermal spring sediments ranging from 21 to 88°C, representing communities of 330 to 3,800 bacterial and archaeal species based on 16S rRNA gene amplicon analysis. Metagenomes were sequenced, and Pfam abundances were used as a proxy for metagenomic functional diversity. Significant decreases in both species diversity and Pfam diversity were observed with increasing temperatures. The relationship between Pfam diversity and species diversity followed a power function with the steepest slopes in the high-temperature, low-diversity region of the gradient. Species additions to simple thermophilic communities added many new Pfams, while species additions to complex mesophilic communities added relatively fewer new Pfams, indicating that species diversity does not approach saturation as rapidly as Pfam diversity does. Many Pfams appeared to have distinct temperature ceilings of 60 to 80°C. This study suggests that temperature stress limits both taxonomic and functional diversity of microbial communities, but in a quantitatively different manner. Lower functional diversity at higher temperatures is probably due to two factors, including (i) the absence of many enzymes not adapted to thermophilic conditions, and (ii) the fact that high-temperature communities are comprised of fewer species with smaller average genomes and, therefore, contain fewer rare functions. IMPORTANCE Only recently have microbial ecologists begun to assess quantitatively how microbial species diversity correlates with environmental factors like pH, temperature, and salinity. However, still, very few studies have examined how the number of distinct biochemical functions of microbial communities, termed functional diversity, varies with the same environmental factors. Our study examined 18 microbial communities sampled across a wide temperature gradient and found that increasing temperature reduced both species and functional diversity, but in different ways. Initially, functional diversity increased sharply with increasing species diversity but eventually plateaued, following a power function. This pattern has been previously predicted in theoretical models, but our study validates this predicted power function with field metagenomic data. This study also presents a unique overview of the distribution of metabolic functions along a temperature gradient, demonstrating that many functions have temperature "ceilings" above which they are no longer found.

Nickel isotopes link Siberian Traps aerosol particles to the end-Permian mass extinction.

The end-Permian mass extinction (EPME) was the most severe extinction event in the past 540 million years, and the Siberian Traps large igneous province (STLIP) is widely hypothesized to have been the primary trigger for the environmental catastrophe. The killing mechanisms depend critically on the nature of volatiles ejected during STLIP eruptions, initiating about 300 kyr before the extinction event, because the atmosphere is the primary interface between magmatism and extinction. Here we report Ni isotopes for Permian-Triassic sedimentary rocks from Arctic Canada. The δ60Ni data range from -1.09‰ to 0.35‰, and exhibit the lightest δ60Ni compositions ever reported for sedimentary rocks. Our results provide strong evidence for global dispersion and loading of Ni-rich aerosol particles into the Panthalassic Ocean. Our data demonstrate that environmental degradation had begun well before the extinction event and provide a link between global dispersion of Ni-rich aerosols, ocean chemistry changes, and the EPME.

GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring.

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria. We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2-79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 105 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45-77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria, representing a candidate order (Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O2, suggesting a microaerophilic lifestyle.

Microbial Metabolic Redundancy Is a Key Mechanism in a Sulfur-Rich Glacial Ecosystem.

Biological sulfur cycling in polar, low-temperature ecosystems is an understudied phenomenon in part due to difficulty of access and the dynamic nature of glacial environments. One such environment where sulfur cycling is known to play an important role in microbial metabolisms is located at Borup Fiord Pass (BFP) in the Canadian High Arctic. Here, transient springs emerge from ice near the terminus of a glacier, creating a large area of proglacial aufeis (spring-derived ice) that is often covered in bright yellow/white sulfur, sulfate, and carbonate mineral precipitates accompanied by a strong odor of hydrogen sulfide. Metagenomic sequencing of samples from multiple sites and of various sample types across the BFP glacial system produced 31 metagenome-assembled genomes (MAGs) that were queried for sulfur, nitrogen, and carbon cycling/metabolism genes. An abundance of sulfur cycling genes was widespread across the isolated MAGs and sample metagenomes taxonomically associated with the bacterial classes Alphaproteobacteria and Gammaproteobacteria and Campylobacteria (formerly the Epsilonproteobacteria). This corroborates previous research from BFP implicating Campylobacteria as the primary class responsible for sulfur oxidation; however, data reported here suggested putative sulfur oxidation by organisms in both the alphaproteobacterial and gammaproteobacterial classes that was not predicted by previous work. These findings indicate that in low-temperature, sulfur-based environments, functional redundancy may be a key mechanism that microorganisms use to enable coexistence whenever energy is limited and/or focused by redox chemistry.IMPORTANCE A unique environment at Borup Fiord Pass is characterized by a sulfur-enriched glacial ecosystem in the low-temperature Canadian High Arctic. BFP represents one of the best terrestrial analog sites for studying icy, sulfur-rich worlds outside our own, such as Europa and Mars. The site also allows investigation of sulfur-based microbial metabolisms in cold environments here on Earth. Here, we report whole-genome sequencing data that suggest that sulfur cycling metabolisms at BFP are more widely used across bacterial taxa than predicted. From our analyses, the metabolic capability of sulfur oxidation among multiple community members appears likely due to functional redundancy present in their genomes. Functional redundancy, with respect to sulfur-oxidation at the BFP sulfur-ice environment, may indicate that this dynamic ecosystem hosts microorganisms that are able to use multiple sulfur electron donors alongside other metabolic pathways, including those for carbon and nitrogen.

Ecological and genomic analyses of candidate phylum WPS-2 bacteria in an unvegetated soil.

Members of the bacterial candidate phylum WPS-2 (or Eremiobacterota) are abundant in several dry, bare soil environments. In a bare soil deposited by an extinct iron-sulfur spring, we found that WPS-2 comprised up to 24% of the bacterial community and up to 108 cells per g of soil based on 16S rRNA gene sequencing and quantification. A single genus-level cluster (Ca. Rubrimentiphilum) predominated in bare soils but was less abundant in adjacent forest. Nearly complete genomes of Ca. Rubrimentiphilum were recovered as single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs). Surprisingly, given the abundance of WPS-2 in bare soils, the genomes did not indicate any capacity for autotrophy, phototrophy, or trace gas metabolism. Instead, they suggest a predominantly aerobic organoheterotrophic lifestyle, perhaps based on scavenging amino acids, nucleotides, and complex oligopeptides, along with lithotrophic capacity on thiosulfate. Network analyses of the entire community showed that some species of Chloroflexi, Actinobacteria, and candidate phylum AD3 (or Dormibacterota) co-occurred with Ca. Rubrimentiphilum and may represent ecological or metabolic partners. We propose that Ca. Rubrimentiphilum act as efficient heterotrophic scavengers. Combined with previous studies, these data suggest that the phylum WPS-2 includes bacteria with diverse metabolic capabilities.

Anomalous fractionation of mercury isotopes in the Late Archean atmosphere.

Earth's surface underwent a dramatic transition ~2.3 billion years ago when atmospheric oxygen first accumulated during the Great Oxidation Event, but the detailed composition of the reducing early atmosphere is not well known. Here we develop mercury (Hg) stable isotopes as a proxy for paleoatmospheric chemistry and use Hg isotope data from 2.5 billion-year-old sedimentary rocks to examine changes in the Late Archean atmosphere immediately prior to the Great Oxidation Event. These sediments preserve evidence of strong photochemical transformations of mercury in the absence of molecular oxygen. In addition, these geochemical records combined with previously published multi-proxy data support a vital role for methane in Earth's early atmosphere.

Natural attenuation of spilled crude oil by cold-adapted soil bacterial communities at a decommissioned High Arctic oil well site.

Romulus C-42 is a decommissioned oil well on Ellesmere Island in the Canadian High Arctic, and is the northernmost well to have produced oil and gas anywhere in the world. The remote site has been untouched since a crude oil spill in 1972, offering a rare opportunity to examine natural attenuation in Arctic soils >40 years after a pollution event. Bacterial community composition in crude oil contaminated soils was significantly different from adjacent background soils. Two members of the genus Rhodanobacter (Alphaproteobacteria) were found consistently in contaminated soils, whereas two members of the genus Sphingomonas (Gammaproteobacteria) appeared opposite to each other, one consistently within the oil contaminated soil and the other consistently in non-oil contaminated soils. GC of soil hydrocarbon extracts revealed moderate levels of biodegradation relative to the original oil produced in 1972. Despite conditions permissive for bacterial activity (>0 °C) being limited to only a few months each year, natural attenuation by cold adapted soil microbial communities has taken place since the oil spill over 40 years ago. Rhodanobacter and Sphingomonas lineages are associated with contaminated and baseline conditions in this extreme environment, revealing the utility of bacterial diversity measurements for assessing long-term responses of Arctic soils to pollution. ORIGINALITY-SIGNIFICANCE STATEMENT: Romulus C-42, the northernmost onshore drilling well in the world, was decommissioned following a small crude oil spill in 1972. Soil bacterial diversity profiles obtained >40 years later revealed significant differences in oil contaminated soils relative to adjacent non-oil contaminated background soils, consistent with evidence for moderate biodegradation of spilled crude oil having taken place since 1972. The results indicate that microbial diversity profiling is an effective tool for assessing natural attenuation in remote High Arctic soils with a history of oil pollution.

Limited freshwater cap in the Eocene Arctic Ocean.

Remains of the freshwater fern Azolla, found in Eocene (~50 Ma ago) sediments in the modern central Arctic Ocean, have been used to suggest that seasonal freshwater caps covered the entire Arctic Ocean during that time, with significant impact on global ocean circulation and climate. However, these records are located on the Lomonosov Ridge, which during the Eocene was a continental fragment barely rifted from Eurasia, separating the smaller Eurasian Basin from the much larger Amerasian Basin to the west. As such, the Lomonosov Ridge does not necessarily record environmental conditions of the broader Arctic Ocean. We tested the hypothesis of freshwater caps by examining sediment records from the western Amerasian Basin. Here we show that in the larger Amerasian Basin the Azolla event is associated with marine microfauna along with allochthonous (terrestrially sourced) organic matter. We propose that Azolla events are related to an increased hydrologic cycle washing terrestrially sourced Azolla, and other organics, into the Arctic Ocean. If freshwater caps did occur, then they were at best restricted to the small Eurasian Basin and would have had a limited impact on Eocene global climate, contrary to current models.

Analysis of microbial communities in natural halite springs reveals a domain-dependent relationship of species diversity to osmotic stress.

Microbial species diversity may peak at certain optimal environmental conditions and decrease toward more extreme conditions. Indeed, bell-shaped relationships of species diversity against pH and temperature have been demonstrated, but diversity patterns across other environmental conditions are less well reported. In this study, we investigated the impact of salinity on the diversity of microorganisms from all three domains in a large set of natural springs with salinities ranging from freshwater to halite saturated. Habitat salinity was found to be linearly and inversely related to diversity of all three domains. The relationship was strongest in the bacteria, where salinity explained up to 44% of the variation in different diversity metrics (OTUs, Shannon index, and Phylogenetic Diversity). However, the relationship was weaker for Eukarya and Archaea. The known salt-in strategist Archaea of the Halobacteriaceae even showed the opposite trend, with increasing diversity at higher salinity. We propose that high energetic requirements constrain species diversity at high salinity but that the diversity of taxa with energetically less expensive osmotolerance strategies is less affected. Declining diversity with increasing osmotic stress may be a general rule for microbes as well as plants and animals, but the strength of this relationship varies greatly across microbial taxa.

Low-Temperature Sulfidic-Ice Microbial Communities, Borup Fiord Pass, Canadian High Arctic.

A sulfur-dominated supraglacial spring system found at Borup Fiord Pass (BFP), Ellesmere Island, Nunavut, Canada, is a unique sulfur-on-ice system expressed along the toe of a glacier. BFP has an intermittent flowing, subsurface-derived, glacial spring that creates a large white-yellow icing (aufeis) that extends down-valley. Over field campaigns in 2014, 2016, and 2017, numerous samples were collected and analyzed for both microbial community composition and aqueous geochemistry. Samples were collected from multiple site types: spring discharge fluid, aufeis (spring-derived ice), melt pools with sedimented cryoconite material, and mineral precipitate scrapings, to probe how microbial communities differed between site types in a dynamic freeze/thaw sulfur-rich system. Dissolved sulfate varied between 0.07 and 11.6 mM and was correlated with chloride concentrations, where the fluids were saltiest among spring fluids. The highest sulfate samples exhibited high dissolved sulfide values between 0.22 and 2.25 mM. 16S rRNA gene sequencing from melt pool and aufeis samples from the 2014 campaign were highly abundant in operational taxonomic units (OTUs) closely related to sulfur-oxidizing microorganisms (SOM; Sulfurimonas, Sulfurovum, and Sulfuricurvum). Subsequent sampling 2 weeks later had fewer SOMs and showed an increased abundance of the genus Flavobacterium. Desulfocapsa, an organism that specializes in the disproportionation of inorganic sulfur compounds was also found. Samples from 2016 and 2017 revealed that microorganisms present were highly similar in community composition to 2014 samples, primarily echoed by the continued presence of Flavobacterium sp. Results suggest that while there may be acute events where sulfur cycling organisms dominate, a basal community structure appears to dominate over time and site type. These results further enhance our knowledge of low-temperature sulfur systems on Earth, and help to guide the search for potential life on extraterrestrial worlds, such as Europa, where similar low-temperature sulfur-rich conditions may exist.

Mercury spikes suggest volcanic driver of the Ordovician-Silurian mass extinction.

The second largest Phanerozoic mass extinction occurred at the Ordovician-Silurian (O-S) boundary. However, unlike the other major mass extinction events, the driver for the O-S extinction remains uncertain. The abundance of mercury (Hg) and total organic carbon (TOC) of Ordovician and early Silurian marine sediments were analyzed from four sections (Huanghuachang, Chenjiahe, Wangjiawan and Dingjiapo) in the Yichang area, South China, as a test for evidence of massive volcanism associated with the O-S event. Our results indicate the Hg concentrations generally vary in parallel with TOC, and that the Hg/TOC ratios remain low and steady state through the Early and Middle Ordovician. However, Hg concentrations and the Hg/TOC ratio increased rapidly in the Late Katian, and have a second peak during the Late Hirnantian (Late Ordovician) that was temporally coincident with two main pulses of mass extinction. Hg isotope data display little to no variation associated with the Hg spikes during the extinction intervals, indicating that the observed Hg spikes are from a volcanic source. These results suggest intense volcanism occurred during the Late Ordovician, and as in other Phanerozoic extinctions, likely played an important role in the O-S event.

Actinocrinis puniceicyclus gen. nov., sp. nov., an actinobacterium isolated from an acidic spring.

An aerobic, mildly acidophilic actinobacterium was isolated from the Ochre Beds bog in Kootenay National Park, Canada. Cells of isolate OB1T were Gram-stain-positive, non-motile, pink- to purple-pigmented filaments. The pH range for growth was pH 3.5-6.5 (optimum pH 5.5), and the temperature range was 13-30°C. The major cellular fatty acids were i-C16 : 0 (28.5 %), i-C15 : 0 (14.6 %) and ai-C15 : 0 (14.3 %), and the major polar lipid was phosphohexose. The major quinone was menaquinone-11 (MK-11), and the peptidoglycan type was A1γ. The DNA G+C content was 70.2 %. Along with growth on complex media including yeast extract, proteose peptone, casamino acids and tryptic soy broth, growth occured on mono- and disaccharides (glucose, sucrose, galactose and xylose) and polysaccharides (starch, gellan, pectin, xylan and alginate). Anaerobic growth was not observed. The cells did not fix atmospheric nitrogen. On the basis of comparative 16S rRNA gene sequence analysis, this isolate belonged to the family Actinospicaceae, in the suborder Catenulisporineae of the order Actinomycetales. The most closely related species was Actinospica robiniae. However, the 16S rRNA gene sequence identity to this bacterium was low (92.8 %) and there were several chemotaxonomic differences from this species. We therefore propose a novel genus and species, Actinocrinis puniceicyclus gen. nov., sp. nov., with strain OB1T (=DSM 45618T=ATCC BAA-2771T) as the type strain.

Global metagenomic survey reveals a new bacterial candidate phylum in geothermal springs.

Analysis of the increasing wealth of metagenomic data collected from diverse environments can lead to the discovery of novel branches on the tree of life. Here we analyse 5.2 Tb of metagenomic data collected globally to discover a novel bacterial phylum ('Candidatus Kryptonia') found exclusively in high-temperature pH-neutral geothermal springs. This lineage had remained hidden as a taxonomic 'blind spot' because of mismatches in the primers commonly used for ribosomal gene surveys. Genome reconstruction from metagenomic data combined with single-cell genomics results in several high-quality genomes representing four genera from the new phylum. Metabolic reconstruction indicates a heterotrophic lifestyle with conspicuous nutritional deficiencies, suggesting the need for metabolic complementarity with other microbes. Co-occurrence patterns identifies a number of putative partners, including an uncultured Armatimonadetes lineage. The discovery of Kryptonia within previously studied geothermal springs underscores the importance of globally sampled metagenomic data in detection of microbial novelty, and highlights the extraordinary diversity of microbial life still awaiting discovery.

Anaerobic carboxydotrophic bacteria in geothermal springs identified using stable isotope probing.

Carbon monoxide (CO) is a potential energy and carbon source for thermophilic bacteria in geothermal environments. Geothermal sites ranging in temperature from 45 to 65°C were investigated for the presence and activity of anaerobic CO-oxidizing bacteria. Anaerobic CO oxidation potentials were measured at up to 48.9 µmoles CO g(-1) (wet weight) day(-1) within five selected sites. Active anaerobic carboxydotrophic bacteria were identified using (13)CO DNA stable isotope probing (SIP) combined with pyrosequencing of 16S rRNA genes amplified from labeled DNA. Bacterial communities identified in heavy DNA fractions were predominated by Firmicutes, which comprised up to 95% of all sequences in (13)CO incubations. The predominant bacteria that assimilated (13)C derived from CO were closely related (>98% 16S rRNA gene sequence identity) to genera of known carboxydotrophs including Thermincola, Desulfotomaculum, Thermolithobacter, and Carboxydocella, although a few species with lower similarity to known bacteria were also found that may represent previously unconfirmed CO-oxidizers. While the distribution was variable, many of the same OTUs were identified across sample sites from different temperature regimes. These results show that bacteria capable of using CO as a carbon source are common in geothermal springs, and that thermophilic carboxydotrophs are probably already quite well known from cultivation studies.

Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil.

The exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced by Gluconacetobacter xylinus or the EPS produced by Beijerinckia indica. The latter is a heteropolysaccharide comprised primarily of l-guluronic acid, d-glucose, and d-glycero-d-mannoheptose. (13)C-labeled EPS and (13)C-labeled cellulose were purified from bacterial cultures grown on [(13)C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from (13)C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However, B. indica EPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylum Planctomycetes. In one incubation, members of the Planctomycetes made up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo- or monosaccharides, the predominance of Planctomycetes suggested that they were primary degraders of EPS. Other bacteria assimilating B. indica EPS included members of the Verrucomicrobia, candidate division OD1, and the Armatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species.

Methanotrophic bacteria in warm geothermal spring sediments identified using stable-isotope probing.

We investigated methanotrophic bacteria in sediments of several warm geothermal springs ranging in temperature from 22 to 45 °C. Methane oxidation was measured at potential rates up to 141 µmol CH4 d(-1) g(-1) sediment. Active methanotrophs were identified using (13) CH4 stable-isotope probing (SIP) incubations performed at close to in situ temperatures for each site. Quantitative (q) PCR of pmoA genes identified the position of the heavy ((13) C-labelled) DNA fractions in density gradients, and 16S rRNA gene pyrotag sequencing of the heavy fractions was performed to identify the active methanotrophs. Methanotroph communities identified in heavy fractions of all samples were predominated by species similar (≥ 95% 16S rRNA gene identities) to previously characterized Gammaproteobacteria and Alphaproteobacteria methanotrophs. Among the five hottest samples (45 °C), members of the Gammaproteobacteria genus Methylocaldum dominated in two cases, while three others were dominated by an OTU closely related (96.8% similarity) to the Alphaproteobacteria genus Methylocapsa. These results suggest that diverse methanotroph groups are adapted to warm environments, including the Methylocapsa-Methylocella-Methyloferula group, which has previously only been detected in cooler sites.

Distribution and diversity of Verrucomicrobia methanotrophs in geothermal and acidic environments.

Recently, methanotrophic members of the phylum Verrucomicrobia have been described, but little is known about their distribution in nature. We surveyed methanotrophic bacteria in geothermal springs and acidic wetlands via pyrosequencing of 16S rRNA gene amplicons. Putative methanotrophic Verrucomicrobia were found in samples covering a broad temperature range (22.5-81.6°C), but only in acidic conditions (pH 1.8-5.0) and only in geothermal environments, not in acidic bogs or fens. Phylogenetically, three 16S rRNA gene sequence clusters of putative methanotrophic Verrucomicrobia were observed. Those detected in high-temperature geothermal samples (44.1-81.6°C) grouped with known thermoacidiphilic 'Methylacidiphilum' isolates. A second group dominated in moderate-temperature geothermal samples (22.5-40.1°C) and a representative mesophilic methanotroph from this group was isolated (strain LP2A). Genome sequencing verified that strain LP2A possessed particulate methane monooxygenase, but its 16S rRNA gene sequence identity to 'Methylacidiphilum infernorum' strain V4 was only 90.6%. A third group clustered distantly with known methanotrophic Verrucomicrobia. Using pmoA-gene targeted quantitative polymerase chain reaction, two geothermal soil profiles showed a dominance of LP2A-like pmoA sequences in the cooler surface layers and 'Methylacidiphilum'-like pmoA sequences in deeper, hotter layers. Based on these results, there appears to be a thermophilic group and a mesophilic group of methanotrophic Verrucomicrobia. However, both were detected only in acidic geothermal environments.